Antimicrobial potential of probiotic cell‐free and Carum copticum L. seed extracts co‐nanoencapsulated in cellulose acetate fibers

Abstract The aim of this work was to co‐nanoencapsulate Lactobacillus acidophilus (LCFE) and Bifidobacterium bifidum (BCFE) cell‐free extract and zenyan (Carum copticum L.) seed water (ZWE) and ethanolic (ZEE) extract in electrospun cellulose acetate (CA) nanofibers and evaluate antimicrobial potential. The zeta potential, SEM image, antibacterial (MIC and MBC), and antifungal (MIC and MFC) activities were evaluated. TPC (total phenol content) of water and ethanol extract of zenyan seed were 14.05 and 136.44 mg GAE/g, respectively. A zeta potential of −40.25, −45.80, −43.71, 48.55, 35.50, 47.93, 31.50, 44.69, and −29.61 mV was found for nanofibers of pure CA (cellulose acetate), CA/LCFE, CA/BCFE, CA/ZWE, CA/ZEE, CA/LCFE/ZWE, CA/LCFE/ZEE, CA/BCFE/ZWE, and CA/LCFE/ZEE, respectively. CA electrospun nanofiber loaded with different extracts showed nanosized diameter and uniform structure. Nanoencapsulated extracts showed considerably higher antibacterial and antifungal activity compared to free extracts. Antibacterial activity of lactobacilli cell‐free extract was higher than bifidobacteria, which indicated the presence of the higher amount of antibacterial compounds in lactobacilli extract. Gram‐positive bacteria (S. aureus and L. monocytogenes) had the lowest MIC and MBC of free and nanoencapsulated extracts while Gram‐negatives (E. coli, S. dysenteriae, and S. enteritidis) had higher MIC and MBC. CA‐coated zenyan extracts (water and ethanolic) inhibited the growth of the assayed fungi at the MIC ranging 0.25 to 0.95%. These concentrations were 1.5–2 times lower than those obtained for pure extracts. For nanoencapsulated cell‐free extracts of both probiotics, the MIC values were about five times lower than the free extracts. The highest antimicrobial activity obtained for CA nanofibers contained zenyan ethanolic extract and cell‐free extract of lactobacilli or bifidobacteria.


| INTRODUC TI ON
The unregulated and extensive consumption of antibiotics to breed and treat livestock and poultry and treat human infections has led to the emergence of antibiotic-resistant microorganisms and concerns about public health (Blair et al., 2015). In the recent decades, many studies have concentrated on the finding and production of natural antimicrobial compounds with high efficacy using new technologies (Liakos et al., 2017;Royo et al., 2010).
Among the antimicrobials of natural origin used in food and pharmaceutical industries, bacterial and plant metabolites are being popular. Probiotic bacteria, mainly bifidobacteria and lactobacilli, as the most used microorganisms in the human diet, produce multiple metabolites like bacteriocins, organic acids, enzymes, fatty acids, vitamins, proteins, peptides, etc. (Saadatzadeh et al., 2013). The species Bifidobacterium animalis colonizes in the mammalian colon and grows considerably in milk and milk-derivate cultures. It shows resistance to low pH and oxidative stress, and its metabolites modulate the immune system and improve gut barrier function (Quigley, 2017). Also, it is reported that B. bifidum as a natural inhabitant of the human gut suppresses the gut inflammation and disturbance resulted from repeated antibiotic therapy (Ojima et al., 2020). The antimicrobial activity of some Lactobacillus species against Enterobacteriaceae and other pathogens is announced too Inglin et al., 2015).
Phenolic compounds are mainly responsible for the antimicrobial and antioxidants activity of herbal extracts and essential oils.
The extensive use of herbal extracts in the food industry to prevent lipid oxidation, retard microbial growth, delay food spoilage, and improve the organoleptic properties of food products has been reported (Parham et al., 2020). Carrum copticum L. (zenyan) of the family Apiaceae is cultivated in many parts of the world such as Iran and India. Traditionally, zenyan seed has been used as a flavoring agent and also for various therapeutic aspects such as respiratory distress, diarrhea, abdominal pain, and abdominal tumors. Several other health benefits including antibacterial, antifungal, and antiparasitic effects have been reported (Lim, 2012;Ramana et al., 2014). Zenyan seed contains important functional compounds like phenolics (carvacrol), thymol, terpinene, paracymene, and beta-pinene (Alavinezhad & Boskabady, 2015;Mahmoudzadeh et al., 2017). The chemical compounds of plant extracts are volatile and easily degrade upon exposure to oxygen, high temperature, and light. Encapsulation and coating techniques such as micro-and nanoencapsulation, micro-and nanoemulsification, producing nanocomplexes, and micro-and nanofibers are used to deliver these sensitive compounds to their specific targets with the minimum loss and controlled release (Azizkhani et al., 2021a;Maes et al., 2019;Prakash et al., 2018;Wadhwa et al., 2017).
Electrospinning is a simple, easy applying, and novel technique to fabricate nanofibers that applies the electric force to draw charged threads of a polymer solution followed by exposure of the fibers to a spinning movement while transferring from a spinneret to a collector plate in the shape of ultrafine nonwoven fiber mats. This method allows producing nanomaterials with desired structural and physicochemical characteristics (Nagy et al., 2014;Xue et al., 2017;Zupančič et al., 2019). One of the biopolymers that can be easily electrospun to ultrafine fibers with nanometer size and possesses good biocompatibility, biodegradability, chemical resistance, and thermal stability is cellulose acetate (CA) (Liakos et al., 2015;Liakos et al., 2017). CA, a natural polymer, is the acetate ester of cellulose and the structural macromolecule of the green plants' cell wall. The objective of the present research was to evaluate the antimicrobial activity of probiotics cell-free and zenyan seed extracts co-nanoencapsulated in electrospun CA nanofibers.

| Measuring total phenolic content of zenyan extract
The total phenolic content (TPC) of zenyan water and ethanol extracts was determined using the Folin-Ciocalteu reagent (Şahin et al., 2013). The working solutions were prepared as follows: solution A contained 2% of aqueous Na 2 CO 3 in 0.1 M NaOH; solution B contained 0.5% of aqueous CuSO 4 in 1% NaKC 4 H 4 O 6 solution; solution C was a mixture of solution A (50 ml) and solution B (1 ml) which was freshly prepared; and Folin-Ciocalteu reagent was made by diluting the stock solution with H 2 O at the ratio of 1:3 (v/v). To conduct the assay, 0.1 ml of the water or ethanol extract was added to 1.9 ml of H 2 O and 2.5 ml of solution C, and this mixture was incubated at ambient temperature for around 10 min. In the next step, 0.25 ml of the Folin-Ciocalteu reagent was added and kept at room temperature for 30 min in order to stabilize its blue color. The solutions' absorbance was read by a spectrophotometer (model Lambda 365; Perkin Elmer, USA) at the wavelength of 750 nm. A standard calibration curve was plotted using multiple concentrations of gallic acid. The TPC was calculated from the standard curve and reported as mg of gallic acid equivalent (GAE) per g of the extract.

| Preparing electrospun nanofibers
The nanoencapsulation process was carried out according to the method of Burgut (2021) with some modifications. Cell-free extracts of L. acidophilus and B. bifidum were encapsulated individually. Co-nanoencapsulation was performed with cell-free extracts of bacteria, zenyan water, or ethanolic extract (1% v/v), and CA (10% w/v) were mixed. The mixtures were homogenized applying an ultrasonic homogenizer for 15 min (Sinosonics, China), and transferred into plastic syringes (attached to 23-gauge stainless steel needles and a syringe pump), and electrospun by a high voltage power supply. The electrospinning process was carried out using a laboratoryscale electrospinner (Vira System, Iran) and optimized to achieve desired nanofibers: different flow rates (1.0 to 6.0 ml/h), voltage values (70 to 150 kV), distances between the Taylor cone and the flat collector (8 to 22 cm), and the combinations of these parameters were tested. The optimization data are as follows: the voltage was adjusted at 125 kV, the flow rate of electrospinning dope solutions was 5 ml/h, and the distance between the needle and aluminum foil as the collector was adjusted at 15 cm. The electrospinning process was performed at 25 ± 1°C and the solutions were volatilized completely during the electrospinning. To remove the remained water, the obtained nanofibers were freeze dried after collecting from the aluminum foil. The average thickness of electrospun nanofiber mats was around 0.18 mm (Liakos et al., 2015;Liakos et al., 2017).

| Zeta potential of nanofiber mats
The zeta potential is indicative of the stability of a colloidal dispersion and the electrophoretic mobility of the particles. The zeta potential of the electrospun nanofiber mats was measured applying the Zetasizer ® Nano ZS (model ZEN 3600, Malvern Instruments, Worcestershire, UK). The samples were prepared through the dispersion of 1 mg of nanofiber mats in 5 ml of PBS and run 10 times at 25 ± 1°C.

| Scanning electronic microscopy (SEM)
To obtain SEM images, one layer of a two-sided tape was attached to the sample place of the scanning electron microscope and the freshly fabricated samples of nanofiber mat were sprayed onto one side of the tape followed by gold spraying. The samples were observed on a high-resolution and low-vacuum scanning electron microscope (MIRA3 FEG-SEM, Tescan Co., Czech).  (Azizkhani et al., 2021b). Briefly, the bacteria E. coli, S. aureus, S. enteritidis, L. monocytogenes, and S. dysenteriae were recovered in the BHI broth at 37 ± 1ºC for 18-24 h, and for each individual bacteria, the cell population was adjusted at 10 6 CFU/ml. The nonencapsulated and nanoencapsulated probiotic and zenyan extracts were diluted, added to the BHI broth, transferred to the microwells up to 180 μl, and then 20 μl of the bacterial inoculum was added and shook horizontally. The microplates were incubated at 37 ± 1ºC for 24 h. MIC was determined as the lowest concentration in which no visible bacterial growth was observed.

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MBC was measured by a subculturing of 50 μl of the wells with no visible growth on the BHI agar after incubating at 37ºC.
The fungi (C. albicans, A. niger, Penicillium sp., and Fusarium sp.) were cultured on the slant potato dextrose agar (PDA) followed by incubation at 30 ± 1°C for 7 days. Then, 10 ml of sterile sodium lauryl sulfate (0.01% w/v in NaCl 1%) was transferred to the slant PDA to prepare monospore suspension. The obtained suspensions were passed through Whatman paper (pore size: 180 μm) and the fungal population was adjusted at 5 × 10 5 conidia/ml. The antifungal experiment was performed on Petri dishes containing malt extract (1% w/v), yeast extract (2% w/v), and agar (2% w/v). After sterilizing and cooling to 45 ± 1ºC, the agar medium was mixed with the extracts (10% v/v) and transferred into the Petri dishes. The plates were inoculated by micropipetting of 10 μl of the conidia suspensions (5 × 10 5 conidia/ml) in the center of the solidified culture medium. The diameter of the inoculums was considered as the initial diameter of the fungal colony. Inoculated Petri dishes were put in plastic boxes (containing bottles of water to prevent dehydration) and incubated at 25 ± 1ºC for 7 days. The diameter of the growth inhibition zone was calculated as: where D control was the mean diameter (mm) of the fungal colony in the control and D sample was the mean diameter (mm) of the antifungaltreated samples.

| Data Analysis
Each experiment was conducted in triplicate and all the statistical analysis was performed by the software of SPSS 22.0 (SPSS Inc., Chicago, IL, USA), using the one-way analysis of variance and the two-sample t-test. The significant differences were determined at the 95% and 99% levels.

| TPC of zenyan extracts
TPCs of water and ethanol extracts of zenyan 14.05 was 136.44 mg GAE/g of the extract, respectively. It is obvious from the results that ethanol extract had significantly higher TPC than water extract (p < .05). Higher TPC would result in higher antimicrobial and other functional activities. In a study by Khanavi et al. (2018), the TPC of ethanol extract of zenyan was 101.7-147.8 mg GAE/g depending on the extraction method (Khanavi et al., 2018). Zenyan mostly consists of monoterpenoids and polyphenols like flavonoids and phenolic acids (Zarshenas et al., 2013).

| Zeta potential
One of the most important and widely used characterization parameters for nanoscaled particles is zeta potential which is an indicator of the surface charge and the electrostatic potential. According to previous studies, well-stabilized nanoparticles with high dispersion constancy showed a zeta potential value of ±30 mV which provides stable suspensions and prevents particle aggregation (Vogel et al., 2017). As presented in Figure 1, negative zeta potential values were found for all nanofibers except for CA loaded with zenyan ethanol extract (p < .05). It was found that the zeta potential of CA/extract nanofiber mats was lower (much negative) than the zeta potential value of pure CA nanofibers (p < .05). The variations in the zeta potential value were attributed to the presence of some compounds and molecules of the extracts on the outer surface of the nanofibers or nanoparticles (Liakos et al., 2018) and the changing manner of the zeta potential in our work shows that the extracts were grafted on the membrane of the CA biopolymer. The zeta potential of CA nanofibers loaded with zenyan ethanol extract was positive, while this value for the nanofibers containing zenyan water extract was found to be negative (p < .05). It is reported that herbal water and ethanol extracts present negative and positive zeta potentials, respectively, which is due to the surface charge of the extract components (Yuwono et al., 2015). Also, there was a slight difference between the zeta potential of the CA nanofibers loaded with cell-free extracts of L.
Our data showed that loading CA nanofibers with probiotic cell-free extracts increased the negativity of the zeta potential in comparison to control (without probiotics) which could be explained   Ji et al., 2019;Murga et al., 2000;Pérez et al., 1998). The changes in the zeta potential value after loading nanofibers with the extracts expressed promising encapsulation of these extracts within CA biopolymer as a nanocarrier.

| SEM images
The images of the control (CA nanofibers without extracts) and extract-loaded electrospun nanofibers obtained by SEM are presented in Figure 2. The diameters of the nanofibers are calculated for 20 fibers for each image and ranged from 255 to 835 nm ( Figure 3). According to the SEM data, CA nanofibers showed uniformity and fracture-free or bead-free morphology with an average diameter of around 285 nm. The CA fibers loaded with different extracts of zenyan and cell-free extracts of bacteria had larger diameters than fibers of pure CA (p < .05). Also, extractloaded fibers showed less uniformity but continuous texture without considerable defect and fracture. From the SEM results, it was observed that loading CA with both zenyan extracts and cell-free extracts of probiotics caused beads to appear in fibers and increased the diameter size (p < .05) but there was no fracture in fibers' structure. In a study by Burgut (2021), the antimicrobial effect of co-microencapsulated lactobacilli cell-free and propolis ethanol and water extracts was evaluated. Wrinkled-shaped microcapsules were found in capsules containing propolis water extracts (Burgut, 2021). In the current research, the absence of fractures and cracks in nanofibers revealed that the encapsulation process was carried out well.
The difference between the diameter size of extract-loaded nanofibers is due to the chemical compounds of the extracts that as bioactive fractions react with hydroxyl moieties of CA and form hemiacetal bonds and uniform morphology or, in contrast, attached to CA as the host polymer and present less uniform and more complex fibers with higher diameters (Lammari et al., 2020).

| Antibacterial activity
The

| Antifungal activity
The antifungal effect of co-encapsulated zenyan water and ethanolic extract with cell-free extract of bifidobacteria and lactobacilli against some fungal food spoiling and human pathogens is presented in  (Kapustová et al., 2021). It is reported that nanocapsules of oregano essential oil showed higher antifungal activity against Penicillium sp., Fusarium sp., and Cladosporium sp. These results are related to the chemical compounds of the extracts. As the main bioactive components of zenyan extracts, particularly ethanolic extract, consisted of polyphenols with high TPC value, their co-encapsulation in CA fibers with cell-free extracts of bifidobacteria and lactobacilli (rich in phenols; organic acids, e.g., acetic acid; hexadecanoic acid; octadecanoic acid; dihydroy benzoic acid; etc.) led to considerable antifungal activity (Burgut, 2021;Nazzaro et al., 2017).

| CON CLUS ION
The CA electrospun nanofibers loaded with water and ethanolic extracts of zenyan and cell-free extracts of L. acidophilus and B. bifidum presented nanosized diameter and almost uniform and defect-free structures. Also, they showed considerably higher antibacterial and antifungal activity compared to free extracts. The highest antimicrobial activity was found for CA nanofibers containing zenyan ethanolic extract and cell-free extract of L. acidophilus and B. bifidum. The results of this work can be extended to food and pharmaceutical industrial experiments to introduce new generation of high-efficiency preservatives. It is suggested that, in addition to industrial research, in vivo studies be conducted to investigate the clinical effects of this type of food additives.

ACK N OWLED G M ENTS
This work has been supported by a research grant from the Amol University of Special Modern Technologies, Amol, Iran.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.